Solution for suppressing deterioration of a separation column for analyzing hemoglobins

ABSTRACT

In the analysis of hemoglobins in blood samples by chromatography, an increase in the pressure in a flow passage line is suppressed, a separation column is made durable against prolonged use by supplying phosphate-based buffer solutions as eluting solutions to a separation column and a solution containing not more than 100 mM of S-(carboxyalkyl)-L-cysteine and phosphate-based buffer agent thereto in the course of a series of analyzing steps.

This is a division of application Ser. No. 08/038,396 filed Mar. 29,1993, now U.S. Pat. No. 5,358,639.

BACKGROUND OF THE INVENTION

1) Field of the Invention

The present invention relates to a method and an apparatus for analyzinghemoglobins, and a solution for suppressing deterioration of aseparation column for use therein, and particularly to techniquessuitable for analyzing hemoglobins in blood samples by liquidchromatography.

2) Related Background Art

It is the conventional practice to analyze glycohemoglobins in bloodsamples by liquid chromatography for diagnosis of diabetes, etc.

Japanese Patent Application Kokai (Laid-open) No. 63-75558 disclosessuch conventional practice, where glycohemoglobins in blood samples areanalyzed by supplying a potassium phosphate buffer solution as aneluting solution to a separation column provided with carboxyl groups asion exchange groups.

Chromatographic separation of blood samples by a phosphate-based buffersolution in a separation column provided with carboxyl groups, etc. asion exchange groups, as in the above-mentioned conventional practice,has the following problem. When operations to separate glycohemoglobins,etc. are repeated in the same liquid chromatographic analyzing apparatusso as to conduct analyzing treatments of a large number of bloodsamples, the pressure in the flow passage line of the analyzingapparatus is gradually increased to lower the separability of samplecomponents. Such an increase in the pressure and decrease in theseparability are due to deterioration of the separation column.

Thus, when the liquid chromatographic analyzing apparatus for analyzingglycohemoglobins, etc. is used for a prolonged time, the deterioratedseparation column must be exchanged with a fresh one. Heretofore,exchange frequency of such deteriorated separation columns was so highthat it caused the operator to do an excess of troublesome work for theexchange with increasing consumption of the separating columns.

SUMMARY OF THE INVENTION

An object of the present inventions is to provide a novel means capableof suppressing an increase in the pressure in the flow passage line inthe analyzing apparatus during chromatography of hemoglobins and ofmaintaining a high component separability for a prolonged time.

Another object of the present invention is to provide a method and anapparatus for analysis capable of suppressing deterioration of aseparation column provided with carboxyl groups or carboxyalkyl groupsas ion exchange groups, and a solution for suppressing columndeterioration.

These objects can be attained by supplying a phosphate-based buffersolution containing not more than 100 mM of S-(carboxyalkyl)-L-cysteineto a separation column when hemoglobins in blood samples are analyzed bysupplying a phosphate-based buffer solution as an eluting solution tothe separation column. The solution can be used as an eluting solutionfor separating blood sample components or as a washing solution(regenerating-solution) for a separation column after the componentseparation.

DETAILED DESCRIPTION OF THE INVENTION

It is known that blood contains hemoglobins, glycohemoglobins,hemoglobin F, etc. as hemoglobin-related substances. The term"hemoglobins" will be used hereinafter to refer to thosehemoglobin-related substances.

A separation column for liquid chromatography for use in the analysis ofhemoglobins is packed with granular fillers with specific exchangegroups bonded to a matrix material. As a matrix material, polymers suchas polyvinyl alcohol, polymethacrylate, etc. or silica are used. As ionexchange groups, carboxyl groups, carboxymethyl groups, etc. are used.

In elution of hemoglobins from a separation column, multi-step elution(usually three-step elution), where a plurality of eluting solutions arestepwise supplied, or gradient elution, where pH of an eluting solutionis gradually changed, is used to shorten the treating time. As aneluting solution, a system using a plurality of solutions containing apotassium phosphate buffer agent (a mixture of potassium dihydrogenphosphate and dipotassium hydrogen phosphate), a system using aplurality of solutions containing a sodium phosphate buffer agent (amixture of sodium dihydrogen phosphate and disodium hydrogen phosphate),or a system using a selected combination of these two former systems isused. Anyway, phosphate-based buffer solutions are used as elutingsolutions.

Any of the eluting solutions is prepared to have a pH of preferably 5.5to 6.5, but a plurality of eluting solutions prepared to have a pH of5.5 to 7.5 can be used, when desired. In case of multi-step elution, thelast step eluting solution is prepared to contain 1 to 10 mM ofS-(carboxyalkyl)-L-cysteine. In case of gradient solution, an elutingsolution containing at least 1 mM of S-(carboxyalkyl)-L-cysteine issupplied to the separation column in the latter half period of theentire elution course. In any case, a solution containing at least 1 mMof S-(carboxyalkyl)-L-cysteine is supplied to the separation columnafter the A1(one)c component of hemoglobins has been eluted from theseparation column.

The solution containing S-(carboxyalkyl)-L-cysteine can be used not onlyas an eluting solution, but also as a column washing solution. In thatcase, the column washing solution is a phosphate-based buffer solutionadjusted to a pH of 5.0 to 6.5, and contains a phosphate-based bufferagent as a main component and 5 to 100 mM ofS-(carboxyalkyl)-L-cysteine.

It is practical to use S-(carboxymethyl)-L-cysteine (which will behereinafter referred to as "S-CMC") as an S-(carboxyalkyl)-L-cysteine.S-(carboxy-ethyl)-L-cysteine can be also used to give the similareffect. For convenience of explanation, description will be madehereinafter, referring only to S-CMC as a typical example ofS-(carboxyalkyl)-L-cysteine. Even if S-CMC is added alone to an elutingsolution or a column washing solution, an effect of suppressingdeterioration of a separation column can be obtained, though dependingon the kind of contamination of the separation column. The effect ofsuppressing the deterioration of a separation column can be much moreenhanced by adding S-CMC to an eluting solution or a column washingsolution together with a surfactant.

As a surfactant to be added to an eluting solution or a washing solutiontogether with S-CMC, a surfactant capable of easily bonding to proteinor lipid is used. For example, polyoxyethylene (10) octyl ether as oneof nonionic surfactants has a strong tendency to bond mainly to protein.Such a surfactant is commercially available from Rohm & Haas Co., USA,as Triton X-100 (trademark). Dodecyl-N-betaine as one of amphotericsurfactants has a strong tendency to bond mainly to lipid. Such asurfactant is commercially available from Kao K.K., Japan as Amphitol24B (trademark).

When blood samples are introduced into a liquid chromatographicanalyzing apparatus and subjected to repeated eluting operations not onthe basis of the present invention, contaminants contained in the bloodsamples, such as protein, lipid, polysaccharides, polynucleotide, etc.are adsorbed onto the surfaces of fillers in the separation column, andare gradually accumulated thereon with increasing number of the bloodsamples introduced. Thus, the function of ion exchange groups of thefillers is gradually deteriorated and the pressure at the inlet side ofthe separation column is also gradually increased. Componentseparability of the separation column is lowered with progressingdeterioration of the separation column, and ultimately the separationcolumn must be exchanged with a fresh one.

When the present invention is applied to liquid chromatography of bloodsamples by supplying a solution containing S-CMC and surfactants as alast step eluting solution, for example, of a three-step elution, to aseparation column, contaminants such as protein, lipid, etc. are hardlyadsorbed onto the surfaces of fillers in the separation column with avery small decrease in the pressure in the flow passage line even if theeluting operations are repeated, and the component separability can bemaintained at a high level for a prolonged time. This means that S-CMCworks as an agent for suppressing deterioration of a separation column.

Still furthermore, a solution containing both S-CMC and surfactantsworks to liberate contaminants once bonded to the filler surfaces andremove them from the filler surfaces, that is, it works to regeneratethe once deteriorated separation column to a reusable state. A solutioncontaining surfactants and not-S-CMC hardly remove the contaminants oncebonded to the filler surfaces. Thus, a solution containing both S-CMCand surfactants can be used as a treating solution for regenerating thedeteriorated separation column.

A solution containing not less than 1 mM of S-CMC has tendency todeteriorate separation of glycohemoglobin components, but a solutioncontaining less than 1 mM of S-CMC has a less effect of suppressingdeterioration of a separation column. Thus, a solution containing notless than 1 mM of S-CMC is supplied to the separation column afterglycohemoglobin components such as A1(one)a, A1(one)b, A1(one)c, etc.have been eluted from the separation column. A solution containing lessthan 1 mM of S-CMC has substantially no adverse effect on the separationof glycohemoglobin components, and thus S-CMC can be added to all theeluting solutions at such a low concentration.

A solution containing more than 10 mM of S-CMC has an adverse effect onthe separation of hemoglobin components (Ao component). Thus, an elutingsolution must be prepared to contain not more than 10 mM of S-CMC. WhenS-CMC is added to a column washing solution, on the other hand, theconcentration can be increased to about 100 mM. In that case, there isno fear of direct influence on the component separation, but it isnecessary to purge the column washing solution from the washedseparation column with a first step eluting solution containing no S-CMCbefore the next blood sample is introduced into the separation column.The higher the concentration of S-CMC, the stronger the action ofdesorbing the contaminants from the filler surfaces and the shorter thetreating time with the column washing solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram outlining the structure of a liquidchromatographic analyzing apparatus according to the present invention.

FIG. 2 is a diagram showing an example of supplying eluting solution forthe analysis of hemoglobins.

FIG. 3A and FIG. 3B are chromatograms effect of the present invention.

PREFERRED EMBODIMENTS OF THE INVENTION

Embodiments of the present invention will be explained below.

Structure of a liquid chromatographic analyzing apparatus for analyzinghemoglobins in blood is outlined in FIG. 1 where three-step elution iscarried out.

In FIG. 1, an eluting solution to be pumped at a predetermined flow rateof 0.5 to 10 ml/min by a feed pump 1 is supplied to a separation column4 through a sampling valve 2 having a constant volume tube 27 and a flowpassage filter 3. A blood sample introduced from the sampling valve 2 isseparated into components in the separation column 4, and the elutedcomponents are detected by a detector 5 comprising anultraviolet-visible light absorption photometer. The thus obtainedchromatogram is displayed on a display unit 7. Peaks of the individualcomponents detected by the detector 5 are subjected to a computationtreatment in a control section 6, and the results of the computationtreatment are displayed with component names, retention time of eachpeak, component concentration of each peak, etc. on the display unit 7.

An eluting solution tank 11 contains a first step eluting solution, aneluting solution tank 12 a second step elution solution and an elutingsolution tank 13 a last step eluting solution. A washing solution tank14 contains a washing solution and is used, when required. In theordinary analyzing operations, eluting steps for each sample, using onlythe eluting solution tanks 11 to 13, are repeated. Individual tanks 11to 14 are provided with valves 16 to 19 correspondingly, and opening orclosing motions of these valves are carried out with timing according toa predetermining program instructed from the control section 6.Activation of feed pump 1, sampling valve 2, detector 5, autosampler 21,etc. is also controlled according to a predetermined program from thecontrol section 6.

An autosampler 21 has a detachable sample rack 22, on which maximum 100sample containers can be placed. As the sample containers, vacuum bloodsampling tubes can be used as such in the ordinary case. The autosamplercomprises a sample injection port 23, a drain port 24, a dilution vessel25, and a washing port 26. The injection port 23 is connected to thesampling valve 2 through a flow passage line 29. The autosampler 21further has a conveying mechanism capable of moving in both directions Xand Y, a pipette nozzle capable of moving freely in the horizontaldirection over the autosampler 21 and moving vertically at a desiredposition by the conveying and a syringe mechanism communicated with thepipette nozzle.

Blood samples are pretreated in the autosample 21 before introductioninto the sampling valve 2. At first, a predetermined amount of a wholeblood sample in one sample container is sampled into the pipette nozzleand discharged into the dilution vessel 25. Successively, the nozzle iswashed by the washing port 26, and a dilution solution is dischargedinto the dilution vessel 25 from the nozzle to dilute the blood to 160times the original volume to uniformly disperse the blood therein. Aportion of the blood sample diluted in the dilution vessel 25 is sampledby the nozzle and fed into the constant-volume tube 27 of the samplingvalve 2 from the sample injection port 23. When the constant-volume tube27 is filled with the blood sample, the sampling valve 2 is switchedover to convey the constant volume of the sample to the separationcolumn 4 by the passing stream of the eluting solution.

The separation column 4 is packed with granular fillers with carboxylgroups or carboxyalkyl groups bonded as ion exchange groups to thematrix material. The separation column 4 has such sizes as an innerdiameter of about 4 to about 6 mm and a length of about 30 to about 80mm. A flow cell in the detector 5 is made of quartz glass and has alight path length of 3 to 10 mm. When an ultraviolet-visible photometeris used as the detctor 5, light having a wavelength of 410 nm isdetected as light for the sample and light having a wavelength of 530 nmis detected as light for reference.

When the time of sample injection is made to be a starting time, elutingsolutions are supplied as shown in FIG. 2, in case of 3-step elutionwithout using a washing solution. That is, an eluting solution B issupplied from the first step eluting solution tank 11, an elutingsolution C from the second step eluting solution tank 12, and an elutingsolution D from the third step eluting solution tank 13. At the startingtime, the eluting solution B is supplied to the separation column 4, and0.6 minutes after the starting time, the eluting solution C is suppliedthereto. 1.6 minutes after the starting time, the eluting solution D issupplied thereto, and 2.0 minutes after the starting time, the elutingsolution B is supplied thereto to make the separation column ready forthe introduction of the next sample. In this example, the time requiredfor one sample is 3.3 minutes, and such eluting solution supplyingoperators are repeated according to the sample-introducing timingprogram.

In the example of FIG. 2, the last step eluting solution D containsS-CMC and a surfactant, and in every sample-introducing cycle, thefillers in the separation column 4 are cleaned by the reagent forsuppressing the deterioration of the separation column 4, as containedin the eluting solution D. The timing for introducing the last stepeluting solution D is just after the A1(one)c component peak has beeneluted from the separation column. Even if supply of the elutingsolution D is started by opening the valve 18 according to instructionsfrom the control section 6 1.6 minutes after the starting time, theeluting solution D starts to flow out of the separation column 4 about 2minutes after the starting time, as shown by dotted line in FIG. 2.

Glycohemoglobin components such as peaks A1(one)a, A1(one)b, A1(one)c,unstable A1(one)c (not shown in the drawing), etc. and hemoglobin Fcomponent as peak F have been already eluted from the separation 1column 4 before the eluting solution D containing S-CMC is supplied tothe separation column. However, peaks Ao as a hemoglobin component areeluted by the eluting solution D.

Examples of the analyzing method will be given below.

EXAMPLE 1

Hemoglobins in blood samples were analyzed by the analyzing apparatus ofFIG. 1. Separation column 4 was packed with fillers having carboxymethylgroups as ion exchange groups. Particle sizes of the fillers were 5 μm.Feed rate of feed pump 1 was set to 1.4 ml/min. Three kinds of elutingsolutions shown in the following Table were used as eluting solutions,and supply of the eluting solution was switched over stepwise accordingto the program as shown in FIG. 2, without using a washing solution.

                  TABLE                                                           ______________________________________                                        Eluting                                                                       solution   Composition         pH                                             ______________________________________                                        1st step   KH.sub.2 PO.sub.4                                                                          50.3   mM    6.23                                     eluting    K.sub.2 HPO.sub.4                                                                          10.7   mM                                             solution   Triton X-100 0.02   wt. %                                                     Amphitol 24B 0.02   wt. %                                          2nd step   KH.sub.2 PO.sub.4                                                                          59.6   mM    6.22                                     eluting    K.sub.2 HPO.sub.4                                                                          12.7   mM                                             solution   Triton X-100 0.02   wt. %                                                     Amphitol 24B 0.02   wt. %                                          3rd step   NaH.sub.2 PO.sub.4                                                                         144    mM    6.20                                     eluting    Na.sub.2 HPO.sub.4                                                                         50.1   mM                                             solution   S-CMC        2      mM                                                        Triton X-100 0.02   wt. %                                                     Amphitol 24B 0.02   wt. %                                          ______________________________________                                    

Comparison was made between a case not based on the present inventionand a case based on the present invention. In the case not based on thepresent invention the same first step eluting solution and the secondstep solution as shown in the foregoing Table were used, whereas thesame third step eluting solution as shown in the foregoing Table, exceptthat no S-CMC was contained therein was used, as the eluting solutions.

Eluting operations were carried out continuously with both groups of theeluting solutions each for 1,000 blood samples. In the case not based onthe present invention, a chromatogram as shown in FIG. 3A was obtained,whereas in the case based on the present invention, a chromatogram asshown in FIG. 3B was obtained.

When 1,000 samples were continuously treated not on the basis of thepresent invention, pressure at the inlet side of the separation column 4was increased to 70 bars from the original 50 bars. As is obvious fromFIG. 3A, separation around the A1(one)b peak was poor and the A1(one)apeak that must have been properly present was not identified.Furthermore, separation around the A1(one)c peak was poor and the peakof unstable A1(one)c (L-A1(one)c) was not identified. That is, only 4components could be identified. Furthermore, the retention time forobtaining the chromatograms had a tendency to become a little shorter atthe later runs than that at the earlier runs.

In the case base on the present invention, the chromatograms obtainedeven after the continuous treatments of 1,000 samples were notsubstantially changed from those obtained at the earlier runs. Pressureat the inlet side of the separation column was increased only to 45 barsfrom the initial 50 bars. As is obvious from FIG. 3B, A1(one)b peak andA1(one)a peak were separated from each other, and A1(one)c peak andlabile A1(one)c peak were separated from each other. That is, the 6components could be identified, and this shows that deterioration of theseparation column was very small. Retention time for obtaining thechromatograms at the later runs was not substantially changed from thatat the earlier runs. It was found that continuous treatments of even10,000 samples could be carried out in the present invention, whilekeeping a high separability.

EXAMPLE 2

To analyze hemoglobins in blood samples in the analyzing apparatus ofFIG. 1, fillers with carboxyl groups as ion exchange groups and havingparticle sizes of 5 μm were packed in the separation column. The firstto third step eluting solutions were buffer solutions each containingdisodium hydrogen phosphate and sodium dihydrogen phosphate and having apH of 5.94 for the first step, a pH of 5.82 for the second step and a pHof 5.71 for the third step. In this example, a column washing solutionwas used besides the eluting solutions.

The washing solution placed in the washing solution tank 14 of FIG. 1was a phosphate based buffer solution containing the same maincomponents as in the eluting solutions, and further containing 20 mM ofS-CMC and 0.5% by weight of Triton X-100 (surfactant) on the basis ofthe solution, adjusted to pH 5.65.

The column washing solution containing S-CMC was supplied to theseparation column 4 after the third step eluting solution, and apredetermined time after the supply the column washing solution wasswitched to the first step eluting solution to obtain a columnequilibrium for the next sample. When the column washing solution wasused besides the eluting solutions, deterioration of the separationcolumn could be suppressed by adding S-CMC and the surfactant in acolumn washing solution, whereby an increase in the pressure of theseparation column subjected to repeated use for a prolonged time couldbe suppressed and a high separability could be maintained, resulting inimproved reproducibility of exact analysis of hemoglobins.

EXAMPLE 3

A separation column packed with fillers with carboxymethyl groups as ionexchange groups and having particle sizes of 7 μm was used, and areagent for suppressing deterioration of the separation columns wasadded to a third step eluting solution as a last step eluting solutionand no column washing solution was used. The first step eluting solutionwas a sodium phosphate-based buffer solution having a pH of 5.92, thesecond step eluting solution was a potassium phosphate-based buffersolution having a pH of 7.25, and the third step buffer solution was asodium phosphate-based buffer solution having a pH of 5.95 andcontaining 5 mM of S-CMC and 0.2% by weight of Triton X-100 on the basisof the solution. In this example, an increase in the pressure of theseparation column could be suppressed in the analysis of hemoglobins.

EXAMPLE 4

Function to regenerate the separability of deteriorated separationcolumn by a reagent for suppressing the deterioration of a separationcolumn was tested. In the separation column, which treated 1,000 bloodsamples not on the basis of the present invention, componentseparability was deteriorated, as shown in FIG. 3A. With the thusdeteriorated separation column, hemoglobins in blood samples wereanalyzed by repeatedly supplying the eluting solutions shown in theforegoing Table to the separation column. Approximately at the 15thcycle after supplying the eluting solutions shown in the foregoing Tablethereto, improvement of component separability could be observed in thechromatograms. Approximately at the 30th cycle, 6 components could beidentified, and approximately at the 100th cycle, substantially the sameseparatability as the original one could be obtained. That is, the samechromatograms as that shown in FIG. 3B could be obtained.

In the present invention, hemoglobins can be analyzed for a prolongedtime while suppressing an increase in the pressure in the flow passageline, and thus suppression of deterioration of a separation column canbe attained with improved reproducibility of analytical results.

What is claimed is:
 1. A solution for suppressing deterioration of aseparation column for analyzing hemoglobins, which comprises 1 to 100 mMof S-(carboxyalkyl)-L-cysteine as a deterioration-suppressing reagentand 0.01 to 1.0% of a surfactant capable of solubilizing contaminantsoriginating from blood samples.
 2. A solution according to claim 1,wherein the solution contains a phosphate-based buffer agent as a maincomponent.
 3. A solution according to claim 2, wherein the solutioncontains 1 to 10 mM of S-(carboxyalkyl)-L-cysteine and is prepared as aneluting solution having a pH of 5.5 to 7.5.
 4. A solution according toclaim 2, wherein the solution contains 5 to 100 mM ofS-(carboxyalkyl)-L-cysteine and is prepared as a column washing solutionhaving a pH of 5.0 to 6.5.